FRNSC 413 Exam 2

• Serology - the medical dealing with serums; in medicine, serology is any lab tests using antigens and antibodies. 
• Serum - the plasma after blood has clotted out of it. As the field has expanded, serologists deal with all types of body fluids; serum = plasma - clotting fixtures
• Antigen - a substance that sparks an immune response in the body (ex: bacteria, virus, pollen, etc.); stimulates the body to produce antibodies (ex: an immune response). On the surface of each cell are millions of cell markers that can function as antigens. Our immune system has the ability to recognize antigens (cells and molecules which do not belong), bind them together and immobilize them. 
• Antibodies/Immunoglobulins (Ig) - produced by B lymphocytes, they are soluble Y-shaped proteins that make up 20% of blood proteins, and will bind bacteria, viruses or large foreign molecules and tag them for destruction.
• Enzyme-Linked Immunosorbent Assay (ELISA) - immobilize the antigen (either directly or to an antibody which is fixed to the plate). The plate is then treated with a solution containing corresponding antibodies that are conjugated with some sort of detection system, usually an enzyme or fluorophore. The color change is proportional to the quantity of antigen present. 
• Enzymes - proteins that work as a catalyst in biological reactions
• Catalyst - speeds up a reaction (ex: potassium permanganate)
• Anabolic Enzyme - builds a product from substrates (ex: DNA polymerase)
• Catabolic Enzyme - breaks down a substrate to form products (ex: lactase)
• Presumptive Testing - indicates that a substance may be present, but not absolutely certain. Catalytic tests depend on a peroxidase-like redox reaction; the iron in hemoglobin can accept electrons, thus the free oxygen is cleaved from the peroxide group of reagents and then reacts with the indicator (colorless → colored). Color catalytic test are very sensitive but not specific. All presumptive tests will produce a false positive reaction under some set of circumstances (won't react with other body fluids, but will react with plant-based enzymes and other substances).
• Leucomalachite Green (LMG) - an acidic presumptive test where for some fabrics, the enhancement fades rapidly (~30 secs.) and cannot be re-enhanced
• • Kastle-Meyer (Phenolphthalein) - a basic presumptive test that is less sensitive and produces fewer false positives
  • Note: any color change occurring after 15-30 seconds may be a false positive 
  • Note: any color change other than pink is not a positive reaction 
  • Hemastix - a one-step presumptive test that was originally designed to be used to detect blood in urine, which makes it a quick and easy presumptive test. When TMB reagent is oxidized, conjugation increases and causes a dark green/blue color change. It is more sensitive than Phenolphthalein, but it also produces more false positives.
• Leucomalachite Green (LMG) - an acidic presumptive test where for some fabrics, the enhancement fades rapidly (~30 secs.) and cannot be re-enhanced; turns blue-green
• Kastle-Meyer (Phenolphthalein) - a basic presumptive test that is less sensitive and produces fewer false positives; turns pink

  • Note: any color change occurring after 15-30 seconds may be a false positive 
  • Note: any color change other than pink is not a positive reaction 
• Hemastix - a one-step presumptive test that was originally designed to be used to detect blood in urine, which makes it a quick and easy presumptive test. When TMB reagent is oxidized, conjugation increases and causes a dark green/blue color change. It is more sensitive than Phenolphthalein, but it also produces more false positives.
• Confirmatory Testing - can conclusively establish the presence of the analyte of interest in a sample. It is less sensitive and requires larger sample sizes for a conclusive result. Some confirmatory tests are presumptive for animal or human blood, and some confirmatory tests do not provide that information. In crystal tests, hemoglobin derivative crystal formation. After confirmation, species determination can be done as needed.
• Hexagon OBTI - hHb reacts with monoclonal anti-human Hb antibodies which are complexed to pink colored particles. The immunocomplex migrates to “T” where captured by an immobilized second antibody directed against hHb forming a pink “T” line. Unreacted reagents migrate further and are bound in a second line by immobilized Immunoglobulin (IgG) antibodies. 
• Takayama Crystal Test (Pyridine-Hemochromogen Test) - the presence of heme produces pink (sometimes red) feathery crystals of pyridine-hemochromogen. The crystals shown here can take up to 30 minutes to form. Due to the time required and the high LOD (often providing inconclusive results), many labs no longer perform the Takayama Test. 
• Innate Immune System: response is quick and non-specific

  • External Barriers: skin and mucous membranes 
  • Mucus Membranes - contains antibacterial proteins, enzymes, and other  chemicals that can destroy bacteria 
  • Internal Defense: inflammation and fever
  • Phagocytes: neutrophils, macrophages, natural killer cells
• Humoral - controlling the fluid; takes down foreign material through neutralization and agglutination.
• B-lymphocytes - full of membrane-bound antibodies; activates upon binding with a foreign object it recognizes 
• Effector Cells - create and release antibodies 
• Memory Cells - maintain the genetic code for specific antibodies
• Effector T-Cells - triggers apoptosis and destroys everything within the infected and dying cell 
• T-lymphocytes - recognize and destroy infected cells 
• Helper T-Cells - call in the immune response via cytokines 
• Memory T-cells - maintains the genetic code for specific antibodies
• Allergies - an overreaction by the immune system to foreign particles that are not generally harmful to individuals (ex: pollen, bee venom, dust, latex, peanuts, etc). The body initiates an immune response when exposed to these particles, even though one is not needed. 
• Fab Domain - region where antibody binds to the antigen 
• Epitope - the structure of the antigen recognized by the antibody of interest 
• Fc Domain - crystallizable fragment; constant and can bind to immune system elements within the body to remove bound antigen 
• Paratope - the complementary structure on an antibody which recognizes and binds to  an antigen
• Antihuman Serum - a mixture with antibodies that react with all of the proteins in human serum and the different regions of each protein. It is produced by injecting human serum into an animal of another species. Most antihuman sera react most strongly with albumin (the most abundant protein). Antihuman sera must be checked for specificity It must not cross-react with sera of common animals, and it must give positive reactions with human fluids. Antiserum can be used for  species testing via precipitation (but we don’t really do that anymore). 
• Antihuman Serum Production:

  1. Inject antigen into rabbit
  2. Antigen activates B cells
  3. Plasma B cells (memory B cells and plasma B cells) produce polyclonal antibodies 
  4. Obtain antiserum from rabbit containing polyclonal antibodies
  5. The serum from an immunized organism contains a collection of antibodies against the antigen (the molecule used for immunization). 
  6. These antibodies bind to the antigen used for immunization. 
  7. Each antibody has its own structure, its own paratope and its own set of cross reactions. 
  8. This heterogeneity makes each antiserum unique
• High Dose Hook Effect (HDHE) - high blood concentration may not produce a positive result due to overwhelmingly high levels of free Hb
• Polyclonal Antibody - antibodies are produced by injecting animals with a specific antigen of choice. As a response, a series of antibodies will be produced in reaction to a variety of different sites on the antigen:

  1. Inject antigen into rabbit
  2. Antigen activates B cells
  3. Plasma B cells (memory B cells and plasma B cells) produce polyclonal antibodies 
  4. Obtain antiserum from rabbit containing polyclonal antibodies
• Monoclonal Antibody - a collection of identical antibodies that interact with a single antigen site (recognizes only one epitope); attack one and only one site of an antigen: 

  1. Immunization: inject antigen into mouse
  2. Fusion: antigen activates B cells and Myeloma cells to produce hybridoma cells 
  3. HAT Selection: hybridoma cells are screened by ELISA for antibody of interest
  4. Antibody produces hybridoma clones and eventually monoclonal antibodies for expansion and stock
• Haptens - half antigens; molecules that are too small. Not every molecule is a suitable antigen If the antigen is too small, the animal’s T-cells will not respond and antibodies will not be produced. The common solution is to link the hapten to a carrier protein, making the target large enough to produce a T-cell response by the immune system. 
• Blood:

  1. Plasma is a solution: substances dissolved in liquid plasma 
  2. Blood is a colloid because it contains suspended solids 
  3. But blood is ALSO a suspension: a solution containing suspended solids which would eventually settle if not in motion
• Plasma - the liquid component (55%) of whole blood; transports substances such as glucose, lipids, hormones, oxygen, clotting factor, waste, etc., and regulated osmotic pressure. Osmotic pressure is a key factor in heart rate and nutrient distribution throughout the body. It’s hard to pump blood around without any pressure. When we take out the platelets from the plasma, we call that SERUM. Serum is used for further testing in the lab. Plasma consists of:

  • 90% water 
  • 8% proteins 
  • Albumin, globulin, fibrinogen 
  • Antibodies, hormones 
  • 2% miscellaneous 
  • Electrolytes 
  • Lipids 
  • Nutrients